antibiotics

Article

Antibiotic Resistance Characteristics ofPseudomonas aeruginosa Isolated from Keratitisin Australia and India

Mahjabeen Khan 1, Fiona Stapleton 1, Stephen Summers 2 , Scott A. Rice 2,3 andMark D. P. Willcox 1,*

1 School of Optometry and Vision Science, UNSW, Sydney, NSW 2052, Australia;mahjabeen.khan@student.unsw.edu.au (M.K.); f.stapleton@unsw.edu.au (F.S.)

2 The Singapore Centre for Environment Life Sciences Engineering (SCELSE), The School of BiologicalSciences, Nanyang Technological University, Singapore 639798, Singapore; ssummers@ntu.edu.sg (S.S.);RSCOTT@ntu.edu.sg (S.A.R.)

3 The ithree Institute, The University of Technology Sydney, Sydney, NSW 2007, Australia* Correspondence: m.willcox@unsw.edu.au; Tel.: +61-2-9385-4164

Received: 2 July 2020; Accepted: 9 September 2020; Published: 14 September 2020�����������������

Abstract: This study investigated genomic differences in Australian and Indian Pseudomonas aeruginosaisolates from keratitis (infection of the cornea). Overall, the Indian isolates were resistant tomore antibiotics, with some of those isolates being multi-drug resistant. Acquired genes wererelated to resistance to fluoroquinolones, aminoglycosides, beta-lactams, macrolides, sulphonamides,and tetracycline and were more frequent in Indian (96%) than in Australian (35%) isolates (p = 0.02).Indian isolates had large numbers of gene variations (median 50,006, IQR = 26,967–50,600) comparedto Australian isolates (median 26,317, IQR = 25,681–33,780). There were a larger number of mutationsin the mutL and uvrD genes associated with the mismatch repair (MMR) system in Indian isolates,which may result in strains losing their efficacy for DNA repair. The number of gene variations weregreater in isolates carrying MMR system genes or exoU. In the phylogenetic division, the number of coregenes were similar in both groups, but Indian isolates had larger numbers of pan genes (median 6518,IQR = 6040–6935). Clones related to three different sequence types—ST308, ST316, and ST491—werefound among Indian isolates. Only one clone, ST233, containing two strains was present in Australianisolates. The most striking differences between Australian and Indian isolates were carriage of exoU(that encodes a cytolytic phospholipase) in Indian isolates and exoS (that encodes for GTPase activatoractivity) in Australian isolates, large number of acquired resistance genes, greater changes to MMRgenes, and a larger pan genome as well as increased overall genetic variation in the Indian isolates.

Keywords: antibiotic susceptibility; WGS; phylogenetic analysis; DNA mismatch repair system

1. Introduction

Pseudomonas aeruginosa is a ubiquitous bacterium which can cause opportunistic or nosocomialinfections in immuno-compromised patients [1]. P. aeruginosa commonly causes corneal (keratitis) [2],respiratory, burn and wound infections, and infections related to medical or surgical devices includingventilator-associated pneumonia [3,4]. P. aeruginosa corneal infections are usually related to contactlens wear, but other risk factors for keratitis in non-contact lens wearers include ocular trauma,ocular surgery, and prior ocular surface disease [5–8].

The prevalence of multi-drug resistant (MDR) or extensively drug resistant strains of P. aeruginosareduces treatment options, significantly increasing morbidity rates [9]. P. aeruginosa is naturallyresistant to some antibiotics due to the possession of specific resistance genes such as catB that confers

Antibiotics 2020, 9, 600; doi:10.3390/antibiotics9090600 www.mdpi.com/journal/antibiotics

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chloramphenicol resistance and an inducible ampC which encodes for a β-lactamase that hydrolysescephalothin and ampicillin, conferring resistance toβ-lactams [10]. Additionally, the regulation of effluxpumps also contributes towards an elevated resistance to antibiotics [11]. For example, expressionof the efflux pump MexAB-OprM contributes towards intrinsic resistance to a broad spectrumof antibiotics [12], whereas the efflux pump MexXY-OprM is involved in the adaptive resistanceto aminoglycosides [13]. Other resistance mechanisms in P. aeruginosa include the acquisition oftransferrable resistance determinants, including those associated with transposons and integrons [14].Antibiotic resistance of P. aeruginosa varies according to the region where the strains have beenisolated [15,16] presumably due to the prescribing practices, availability of antibiotics, and perhapstheir use in animal husbandry. Various epidemiological studies have identified MDR P. aeruginosa fromdifferent infections and these isolates have acquired different resistance characteristics. For example,aminoglycoside resistance [17] and ciprofloxacin persistence [18] are found in cystic fibrosis isolates ofP. aeruginosa. Some of these MDR strains are clonal and such clonal strains are often the predominantglobal clinical MDR isolates [19] which spread resistance characteristics into the wider populationwhich enables clonal lineages to expand with time.

ExoU has been associated with virulence of P. aeruginosa at the ocular surface. ExoU is aphospholipase that causes mammalian cell death [20] and exoU possession is common in strainsisolated from ocular infections [21]. There is a correlation between carriage of exoU and elevatedresistance to fluoroquinolones and aminoglycosides [22]. ExoU is carried by strains on a genomicisland that also contains resistance genes for a range of antibiotics [23].

In addition to the acquisition of resistance genes, bacteria can develop resistance through mutationof genes so that antibiotic targets are modified. Mutation rates are elevated in strains that carrymutations in DNA mismatch repair (MMR) systems and hence such mutator strains will normally carrymore mutations than non-mutator strains [24]. In P. aeruginosa, the MMR system is composed of mutS,mutL, and UvrD genes [25]. Strains of P. aeruginosa isolated from the lungs of cystic fibrosis patientshave alterations in the DNA MMR system and this has been correlated with multiple antimicrobialresistance [23].

In Australia, there is a tight regulation of prescribing antibiotics, and antibiotics can only beobtained legally with a prescription from a qualified healthcare professional according to the TherapeuticGoods Act 1989. In India, on the other hand, whilst branded antibiotics exist, other forms such ascounterfeit, substandard, and ‘spurious’ antibiotics have been reported [26], making surveillance andregulation difficult [27]. While the antibiotic consumption per person in Australia and India in 2010was approximately similar, there was a more rapid increase between 2000 and 2010 in India [28].These differences may affect antibiotic resistance development.

The aim of the current study was to compare the phenotypic resistance and genetic characteristicsassociated with resistance between strains isolated from Australia and India to better understand theunderlying factors that may lead to an increased resistance in P. aeruginosa strains associated withocular infection.

2. Results

2.1. Antibiotic Susceptibility

The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs)of the P. aeruginosa isolates were determined (Table 1). Strains showing intermediate resistance (I)as well as full resistance to antibiotics were categorized as resistant (R) for subsequent analyses.Based on the Centers for Disease Control and Prevention’s (CDC, Atlanta, GA, USA) definition ofmulti-drug resistance as “an isolate that is resistant to at least one antibiotic in three or more drugclasses”, isolates 198, 202, 216, 217, 218, 219, 220, and 221 were deemed to be multi-drug resistant.Australian isolates 223, 224, 225, 227, 233, and 235 were also resistant to three antibiotics but theseantibiotics were not of different classes. Isolates 176, 193, and 206 were sensitive to all antibiotics, but all

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other isolates were resistant to at least one antibiotic. Overall, Indian isolates were more resistant toantibiotics compared to Australian isolates. Among Australian isolates (n = 14), resistance was 78%for imipenem, 57% for ceftazidime, 50% for ciprofloxacin, 21% for piperacillin, 14% for levofloxacin,7% for tobramycin, and no isolates were resistant to gentamicin or polymyxin. In contrast, resistance inIndian isolates (n = 12) was 75% for ciprofloxacin, 58% for imipenem, 50% for levofloxacin, tobramycin,and ceftazidime, 41% for piperacillin, 40% for gentamicin, and 25% for polymyxin.

Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) ofantibiotics to Pseudomonas aeruginosa keratitis isolates.

StrainNumber Fluoroquinolones * Aminoglycosides β-Lactams

Poly-Peptide2nd

Generation3rd

GenerationPenicillin 4thGeneration Carba-Penem Cephalosporin

3rd Generation

Cipro µg/mL≤1, 2, ≥4 #

Levo µg/mL≤2, 4, ≥8

Genta µg/mL≤4, 8, ≥16

Tobraµg/mL≤4, 8, ≥16

Pipera µg/mL≤16

Imi µg/mL≤2, 4, ≥8

Ceftaz µg/mL≤8, 16, ≥32

PMB µg/mL≤2, 4, ≥8

MIC/MBC

MIC/MBC

MIC/MBC

MIC/MBC

MIC/MBC

MIC/MBC

MIC/MBC

MIC/MBC

123 1/1 1/1 0.25/0.5 4/4 8/16 4(I)/8 2/2 1280(R)/1280126 0.5/1 0.5/1 0.5/1 0.25/0.5 8/16 8(R)/16 128(R)/256 1/1127 1/2 0.25/1 2/4 32(R)/128 4/16 4(I)/8 128(R)/256 0.5/1162 0.5/1 0.5/1 0.25/0.5 0.25/1 8/8 4(I)/4 2/4 0.25/0.5169 2(I)/4 0.25/0.5 0.25/0.5 0.25/0.5 4/8 2/4 1/2 0.25/0.25176 0.5/1 0.25/0.5 0.25/0.5 0.25/0.5 4/8 2/8 2/4 0.25/0.5181 1/4 0.25/0.5 0.25/0.5 0.25/0.5 32(R)/64 4(I)/8 16(I)/32 0.5/1182 1/2 0.25/0.5 0.25/0.5 0.25/0.5 4/8 8(R)/16 1/2 0.25/0.5223 64(R)/128 1/2 0.5/1 0.5/1 160(R)/320 1/2 16(I)/32 2/4224 16(R)/32 1/2 0.25/0.5 0.25/0.5 8/16 64(R)/128 16(I)/32 1/2225 64(R)/128 16(R)/32 0.5/2 1/2 16/32 64(R)/128 8/16 0.25/0.5227 64(R)/128 64(R)/128 0.5/1 0.25/1 16/32 16(R)/32 16(I)/32 0.25/0.5233 8(R)/16 1/2 1/2 105/1 16/32 4(I)/8 160(I)/320 0.5/1235 16(R)/32 0.5/1 2/4 0.5/1 64(R)/128 4(I)/8 64(I)/128 0.25188 2(I)/4 1/2 0.5/1 32(R)/64 16/65 0.5/1 4/8 2/4

189 0.25/1 1/2 0.25/0.5 16(R)/32 4/8 2/1 8/16 2/4

193 1/1 0.25/1 0.25/0.25 0.25/0.5 4/8 2/4 2/2 0.5/1

198 1280(R)/2560 320(R)/1280 2560(R)/5120 16(R)/16 8/8 1/2 8/8 4(I)/4

202 640(R)/1280 320(R)/640 8(I)/32 320(R)/640 16/64 8(R)/32 8/32 0.25/0.25

206 1/1 0.5/0.5 1/1 0.25/0.5 8/8 2/4 2/4 0.25/0.5

216 64(R)/128 4 (I)/8 1/2 0.5/2 160(R)/320 16(R)/32 64(R)/128 64(R)/128

217 64(R)/128 32(R)/64 1/2 1/2 64(R)/128 8(R)/16 32(R)/64 0.25/1

218 8(R)/16 1/2 0.5/1 0.5/1 160(R)/320 8(R)/16 64(R)/128 1/4

219 ≥5120(R)/≥5120 640(R)/1280 ≥5120(R)/≥5120 1280(R)/2560 2560(R)/5120 40(R)/80 16(I)/32 0.25/1

220 2(I)/4 0.25/0.5 0.5/1 0.5/1 0.25/0.5 8(R)/16 160(R)/320 8(R)/16

221 2560(R)/5120 2560(R)/5120 2560(R)/5120 2560(R)/5120 64(R)/128 16(R)/32 32(R)/64 0.25/1

Data for Australian isolates (shaded in gray). Data for 123–182 is from a previously published study [29]. Strains 188–221were Indian keratitis isolates. R = resistant, I = intermediate resistance. * Cipro = Ciprofloxacin, Levo = Levofloxacin,Genta = Gentamicin, Tobra = Tobramycin, Pipera = Piperacillin, Imi = Imipenem, Ceftaz = Ceftazidime,PMB = Polymyxin B; # = Antibiotic breakpoints for sensitive, intermediate, resistant classifications.

2.2. General Features of the Genomes

The isolates after de novo assembly consisted of different numbers of contigs ranging from 50 forisolate 169 to 1917 for isolate 216. The average number of coding sequences was 6162 ± 359.2 forthe Australian isolates and 6544 ± 889 for the Indian isolates. Isolates had an average of 66.1% G +

C content. The tRNA copy number for the isolates ranged from 57 to 86 (which may vary betweenstudies that use different assembly methods). The general features of the isolates are provided inSupplementary Table S1.

2.3. Acquired Resistance Genes

P. aeruginosa isolates were examined for horizontally acquired antibiotic resistance genes (Table 2)using the Resfinder database. Altogether, 33 different acquired antibiotic resistance genes for various

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classes of antibiotics including aminoglycosides, fluoroquinolones, beta-lactams were found in theseisolates (Table 2).

Table 2. Acquired resistance genes in P. aeruginosa isolates from India and Australia.

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Table 2. Acquired resistance genes in P. aeruginosa isolates from India and Australia.

Genes Australian Isolates Indian Isolates

123 126 127 162 169 176 181 182 223 224 225 227 233 235 188 189 193 198 202 206 216 217 218 219 220 221 Aminoglycoside resistance genes

aph(3’)-IIb aph(6)-Id l; rmtD2 rmtB aph(3’)-VI aph(3’)-lIb aph(3’’)-Ib aac(6’)-Ib3 aac(3)-IId aadA1 aac(6’)-Ib-cr

Fluoroquinolone resistance genes crpP qnrVC1

Beta-lactamase resistance genes blaPAO blaLCR-1 blaOXA-485 blaOXA-486 blaOXA-488 blaOXA-396 blaOXA-395 blaOXA-50 blaOXA-10 blaTEM-1B blaVIM-2 blaPME-1 blaPAU-1

Sulphonamide, tetracycline, macrolide, fosfomycin, and chloramphenicol resistance genes sul1 tet(G) mph(E) mph(A) msr(E)* fosA catB

* msr(E) encodes macrolide and lincosamide resistance. Isolates shaded in grey indicate Australian strains. Black color represents gene presence.

Several types of non-synonymous variations were found in the core genome of the keratitis P. aeruginosa isolates when compared with the reference genome of PAO1 (Table 3). These non-synonymous mutations included single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs), deletions, insertions, and complex variations (where more than one change occurred at one specific location compared to the reference strain). The total variations in the isolates ranged from 76,080 in isolate 206 to 22,536 in isolate 181. There was a median of 26,317 (IQR = 25,681–33,780) variations in the genomes of Australian isolates and a median of 50,006 (IQR = 26,967–50,600) in the Indian isolates (p = 0.09). Based on the grouping of core genome phylogeny, isolates within group 2 (198, 202, 219, 220, 221, 233) had the most variations. Isolate 206, which had a unique sequence type and was placed in a separate group by pan genome analysis, had an exceptionally high number of variations (76,080) and SNPs (67,271).

Table 3. Frequency of different types of variation in the genes of P. aeruginosa isolates.

* msr(E) encodes macrolide and lincosamide resistance. Isolates shaded in grey indicate Australian strains. Black colorrepresents gene presence.

An aminoglycoside resistance gene (aph(3’)-IIb), a beta-lactam resistance gene (blaPAO), a fosfomycinresistance gene (fosA), and a chloramphenicol resistance gene (catB7) were common to all isolates.The Australian isolates (123–182) had acquired only eight resistance genes, while the Indian isolates(188–221) had acquired 26 different resistance genes (Table 2). Five Indian isolates (198, 202, 217, 219,and 221, with large pan genomes) acquired the largest number of resistance genes. Of these five isolates,the pairs 198/219 and 202/221 had the most similar resistance gene profiles and each member of thepair were of the same sequence type, ST308 and ST316 respectively. As acquired resistance genes maybe carried on integrons, the genomes of the P. aeruginosa isolates were analyzed for integrons using

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Integron Finder version 1.5.1. qnrCV1 was associated with a class 1 integron in isolates 202 and 221 anda Tn3 transposon in isolates 198 and 219.

Several types of non-synonymous variations were found in the core genome of the keratitisP. aeruginosa isolates when compared with the reference genome of PAO1 (Table 3). These non-synonymousmutations included single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs),deletions, insertions, and complex variations (where more than one change occurred at one specificlocation compared to the reference strain). The total variations in the isolates ranged from 76,080 inisolate 206 to 22,536 in isolate 181. There was a median of 26,317 (IQR = 25,681–33,780) variations inthe genomes of Australian isolates and a median of 50,006 (IQR = 26,967–50,600) in the Indian isolates(p = 0.09). Based on the grouping of core genome phylogeny, isolates within group 2 (198, 202, 219, 220,221, 233) had the most variations. Isolate 206, which had a unique sequence type and was placed in aseparate group by pan genome analysis, had an exceptionally high number of variations (76,080) andSNPs (67,271).

Table 3. Frequency of different types of variation in the genes of P. aeruginosa isolates.

P. aeruginosaIsolates

TotalVariants

VariantComplex

VariantsInsertions

VariantsDeletions

VariantsMNP Variant SNP

123 28,279 1593 187 163 398 25,938126 26,258 1416 164 159 355 24,164127 25,760 1362 163 176 391 23,668162 50,999 3481 281 257 951 46,029169 50,283 3359 269 245 922 45,488176 26,065 1372 168 161 342 24,022181 22,536 1063 162 133 283 20,895182 25,684 1359 172 180 368 23,605223 25,672 1358 167 176 402 23,568224 26,376 1435 163 165 353 24,260225 28,070 1566 167 156 385 25,796227 28,000 1560 162 154 370 25,754233 52,392 3590 285 263 956 47,298235 24,919 1349 162 171 354 22,883188 25,833 1435 164 154 351 23,729

189 25,910 1458 165 155 365 23,767

193 26,567 1445 180 147 389 24,406

198 50,631 3503 280 236 945 45,667

202 49,981 3461 257 236 902 45,125

206 76,180 6449 336 371 1653 67,271

216 28,166 1548 183 164 433 25,838

217 51,119 3575 290 226 944 46,084

218 29,161 1676 182 181 430 26,692

219 50,507 3484 273 237 925 45,588

220 50,180 3452 267 234 894 45,332

221 50,030 3477 260 237 906 45,150

SNP = single nucleotide polymorphism; MNP = multi-nucleotide polymorphism. Isolate numbers highlighted ingray are from Australia.

Non-synonymous mutations were assessed in resistance genes of the P. aeruginosa isolates(Supplementary Table S2). There were no large differences in the mutations in resistance genesof any of the isolates except the antibiotic efflux-related genes opmH and rosC. opmH had ≥9 mutations inall isolates except 127, 162,169, 202, 218, 220, and 221 (mostly isolates of group 2 of core and pan genomephylogenies except 127 and 218). rosC had 20 non-synonymous mutations including insertions/deletions

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in isolate 206, 11 in 233, and ≥5 mutations in isolates 162, 169, 176, 202, 216, 217, 219, 220, and 221(mostly isolates of group 2 of core and pan genome phylogenies except 176, 216), but ≤3 mutations inisolates 123, 126, 123, 181, 182, 188, 189, 193, 198, and 218 (mostly isolates of group 1 of core and pangenome phylogenies except 198). Mutations in efflux genes encoding efflux pumps were also found,including mexX, mexT, mexD, mexM, and mexY, although there was no significant difference betweentwo groups in the possession of mutations in these genes. All other mutations in the genes were randomwithout any association to sequence type, phylogeny, or susceptibility to antibiotics.

2.4. Possession of exoU and Mutations in the DNA Mismatch Repair System

ExoU was present in the genomes of all isolates in group 2 (core and pan genome phylogeneticgroup) as well as isolates 123 and 127. All other isolates possessed exoS with the exception of isolate 126which possessed both exoU and exoS. To address differences in the numbers of sequence variants betweenthe isolates, the genes involved in the DNA mismatch repair (MMR) system mutS (that encodes a proteinwhich binds to errors in DNA), mutL (that encodes a protein that works in synergy with MutS andactivates UvrD), and uvrD (a DNA helicase active in DNA replication) were examined. The mutationsin the MMR system included SNPs, indels, and complex variants. The number of mutations in mutLranged from 1 to 2 and mutations in mutS (which ranged between 0 and 2) were found in seven isolates(Table 4). In uvrD, the number of mutations ranged between 0 and 5 (Table 4). exoU containing isolatespossessed a median of two (IQR = 1–3) mutations in mutL, zero (IQR = 0–2) mutations in mutS, and four(IQR = 2–5) mutations in uvrD, whereas exoS containing isolates possessed a median of zero (IQR = 0–1)mutations in mutL, zero (IQR = 0–1) mutations in mutS, and two (IQR = 0–2) median mutations in uvrD.There were significant differences in the number of mutL (p = 0.0021) and uvrD (p = 0.02) mutations inexoS and exoU isolates but not with mutS (p = 0.3). Isolate 206, an exoS strain and an outlier in the coregenome analysis, had one mutation in mutL. Details of mutations occurring in nucleotide and respectiveproteins are provided in Supplementary Table S3.

Table 4. Possession of exoU and exoS and number and type of non-synonymous mutations in themismatch repair system genes in P. aeruginosa isolates.

P. aeruginosa Isolates Type III SecretionSystem Genes mutL mutS uvrD

123 exoU 1 SNP 0 1 complex126 exoU/exoS 0 0 0127 exoU 0 1 MNP 1 MNP, 1 complex162 exoU 1 SNP 0 2 SNP, 1 MNP, 2 complexes169 exoU 1 SNP 1 complex 2 SNP, 2 MNP, 1 complex176 exoS 1 SNP 0 1 SNP181 exoS 0 0 0182 exoS 0 0 1 MNP 1complex223 exoS 0 1 SNP 1 MNP, 1 complex224 exoS 1 SNP 0 1 MNP, 1 complex225 exoS 0 0 2 SNP, 2 MNP, 1 complex227 exoS 0 0 2 SNP, 2 MNP, 1 complex233 exoU 0 0 1 MNP, 1 complex235 exoS 0 0 0188 exoS 0 0 1 MNP, 1 complex

189 exoS 1 SNP 0 1 MNP, 1 complex

193 exoS 0 0 0

198 exoU 2 SNP 0 1 SNP, 3 complexes

202 exoU 1 SNP 1 complex 1 SNP, 2 MNP, 2 complexes

206 exoS 1 MNP 1 complex 0

216 exoS 0 0 0

217 exoU 2 SNP 1 complex 1 SNP, 2 MNP, 1 complex

218 exoS 0 0 0

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Table 4. Cont.

P. aeruginosa Isolates Type III SecretionSystem Genes mutL mutS uvrD

219 exoU 2 SNP 0 1 SNP, 1 MNP, 2 complexes

220 exoU 1 SNP 1 complex 1 SNP, 2 MNP, 2 complexes

221 exoU 1 SNP 1 complex 1 SNP, 2 MNP, 2 complexes

SNP = single nucleotide polymorphism, MNP = multinucelotide polymorphism. Isolates shaded in grey indicateAustralian strains.

2.5. Sequence Type Analysis and Phylogenetics

All Australian isolates were of different sequence types (ST), except 225 and 227 which belonged toST233. Among the 12 Indian isolates, one isolate was designated as belonging to a new sequence type,two isolates (198 and 219) belonged to ST308, two others (188 and 189) belonged to ST491, and threeisolates (202, 220, and 221) belonged to ST316 (Table 5).

Table 5. Sequence types of P. aeruginosa isolates.

P. aeruginosa Isolates Sequence Types Core Genes Shell Genes Pan/Total Genes123 ST218 5496 508 6004126 ST2726 5483 712 6195127 ST845 5483 938 6421162 ST298 5439 905 6344169 ST1027 5456 694 6150176 ST709 5547 1112 6659181 ST244 5588 1047 6662182 ST27 5486 1096 6582223 ST17 5471 1232 6703224 ST168 5483 607 6090

225 ¤ ST233 5515 1338 6853227 ¤ ST233 5493 1304 6797233 NEWST 5440 624 6064235 ST262 5470 540 6010

188 * ST491 5490 535 6025

189 * ST491 5492 531 6023

193 ST760 5490 594 6084

198 † ST308 5454 1428 6882

202 # ST316 5425 1505 6930

206 NEWST 5331 1084 6415

216 ST1527 5480 1488 6968

217 ST1047 5448 1173 6621

218 ST3083 5513 488 6001

219 † ST308 5451 1796 7247

220 # ST316 5430 948 6378

221 # ST316 5425 1511 6936

PA7 ST1196 3599 4586 8185

PA14 ST253 5436 790 6226

Gray shading denotes Australian isolates. *, †, #, ¤ indicates strains belong to the same sequence types (STs).

The number of core and total or pan (or total) genes were reported from the statistical summaryof Roary v3.11.2. The core genomes of the isolates were aligned using PA7 (Accession numberNC_009656.1), PA14 (Accession number NC_004863.1), and PAO1 (Accession number NC_002516.1) as

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reference strains. The eight published genomes of P. aeruginosa isolates from eye as well as strains fromother sources were also included. The core genes of published isolates are provided in SupplementaryTable S4. The isolates were sub-grouped based on the number of core genes; isolates with a similarnumber of core genes were closely aligned and isolates with the same sequence type were groupedtogether. The core genomes formed two groups in the phylogenetic tree (Figure 1). Isolates in group 1tended to have a larger number of core genes than isolates in group 2. Isolate 206, PA57, and PA7 wereoutliers based on core genome phylogeny. The Australian and Indian isolates had a similar number ofcore genes, whereas the Indian isolates had a larger number of pan genes (10,889) due to the acquisitionof shell genes (genes present in two or more strains) (Table 4).

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Gray shading denotes Australian isolates. *, †, #, ¤ indicates strains belong to the same sequence types (STs).

The number of core and total or pan (or total) genes were reported from the statistical summary of Roary v3.11.2. The core genomes of the isolates were aligned using PA7 (Accession number NC_009656.1), PA14 (Accession number NC_004863.1), and PAO1 (Accession number NC_002516.1) as reference strains. The eight published genomes of P. aeruginosa isolates from eye as well as strains from other sources were also included. The core genes of published isolates are provided in Supplementary Table S4. The isolates were sub-grouped based on the number of core genes; isolates with a similar number of core genes were closely aligned and isolates with the same sequence type were grouped together. The core genomes formed two groups in the phylogenetic tree (Figure 1). Isolates in group 1 tended to have a larger number of core genes than isolates in group 2. Isolate 206, PA57, and PA7 were outliers based on core genome phylogeny. The Australian and Indian isolates had a similar number of core genes, whereas the Indian isolates had a larger number of pan genes (10,889) due to the acquisition of shell genes (genes present in two or more strains) (Table 4).

Figure 1. Core genome phylogeny of P. aeruginosa isolates using Parsnp. PAO1 was used as reference. PA7 and PA14 were also included.

The phylogenetic relationships of these P. aeruginosa isolates were assessed by aligning their pan genome against PAO1 as a reference. The output generated using Roary showing the gene presence or absence in all isolates is provided in Supplementary Figure S1. This again divided the P. aeruginosa isolates into two major groups. Six multi-drug resistant Indian isolates (198, 202, 217, 219, 220, 221) and the VRFPA04 isolate (isolated from the cornea) were clustered in one group, which also

Group 1

Group 2

Figure 1. Core genome phylogeny of P. aeruginosa isolates using Parsnp. PAO1 was used as reference.PA7 and PA14 were also included.

The phylogenetic relationships of these P. aeruginosa isolates were assessed by aligning their pangenome against PAO1 as a reference. The output generated using Roary showing the gene presence orabsence in all isolates is provided in Supplementary Figure S1. This again divided the P. aeruginosaisolates into two major groups. Six multi-drug resistant Indian isolates (198, 202, 217, 219, 220, 221) andthe VRFPA04 isolate (isolated from the cornea) were clustered in one group, which also contained thetwo Australian isolates 162 and 169. The Indian isolate 216 was categorized in a separate sub-groupdue to the large number of shell genes and possession of exoS.

The second group (group 2 of the pan genome analysis) included most of the Australian (123, 126,127, 162, 176, 181, 182, 223, 224, 225, 227, 235) and Indian (188, 189, 193, 216 218) isolates along withreference strain PAO1 (Figure 2). Overall, the multi-drug resistant Indian isolates had a large pangenome (total of 10,889 genes obtained from the statistical summary in Roary v3.11.2). The pan genomegrouping of isolates was broadly based on the number of pan (or total) genes and possession of either

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exoU or exoS in each group, except two Australian isolates 123 and 127 which were in group 2 butpossessed exoU. The other exception to this grouping pattern was for isolates 181 and 182 which hadlarge pan genomes and were clustered into group 1 but carried exoS.

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contained the two Australian isolates 162 and 169. The Indian isolate 216 was categorized in a separate sub-group due to the large number of shell genes and possession of exoS.

The second group (group 2 of the pan genome analysis) included most of the Australian (123, 126, 127, 162, 176, 181, 182, 223, 224, 225, 227, 235) and Indian (188, 189, 193, 216 218) isolates along with reference strain PAO1 (Figure 2). Overall, the multi-drug resistant Indian isolates had a large pan genome (total of 10,889 genes obtained from the statistical summary in Roary v3.11.2). The pan genome grouping of isolates was broadly based on the number of pan (or total) genes and possession of either exoU or exoS in each group, except two Australian isolates 123 and 127 which were in group 2 but possessed exoU. The other exception to this grouping pattern was for isolates 181 and 182 which had large pan genomes and were clustered into group 1 but carried exoS.

Figure 2. Pan genome phylogeny of P. aeruginosa isolates. Branches with no color representation indicate non-ocular isolates. Red color indicates Australian, blue color represents Indian, and orange color represents published eye isolates. Green color represents reference strains. Purple color represents reference strains.

The isolates of group 2 usually had a large number of pan genes and were exoU+. Isolates having similar numbers of pan genes were sub-grouped together. For example, isolate 193 (pan genes = 6084) and 218 (pan genes = 6001) were sub-grouped together. Isolate 218 had a similar number of pan genes to isolate 123 (pan genes = 6001), but isolate 218 possessed exoS, while 123 possessed exoU, and thus these were not grouped together. Isolates belonging to the same sequence type were also grouped together. The MDR isolates, the isolates with same STs, and isolates with large gene variations were clustered in one pan-group. The previously published isolates PA_D1, PA_D2, PA_D9, and PA_D16

Group 1

Group 2

Figure 2. Pan genome phylogeny of P. aeruginosa isolates. Branches with no color representationindicate non-ocular isolates. Red color indicates Australian, blue color represents Indian, and orangecolor represents published eye isolates. Green color represents reference strains. Purple color representsreference strains.

The isolates of group 2 usually had a large number of pan genes and were exoU+. Isolates havingsimilar numbers of pan genes were sub-grouped together. For example, isolate 193 (pan genes = 6084)and 218 (pan genes = 6001) were sub-grouped together. Isolate 218 had a similar number of pan genes toisolate 123 (pan genes = 6001), but isolate 218 possessed exoS, while 123 possessed exoU, and thus thesewere not grouped together. Isolates belonging to the same sequence type were also grouped together.The MDR isolates, the isolates with same STs, and isolates with large gene variations were clustered inone pan-group. The previously published isolates PA_D1, PA_D2, PA_D9, and PA_D16 with the sameST and those with large shell genes were grouped with the MDR isolates of the current study.

3. Discussion

This study investigated genomic differences in Australian and Indian P. aeruginosa isolates fromkeratitis. Phenotypically, more resistance was found in Indian isolates compared to Australian isolatesas has been shown in previous studies [30,31]. Unregulated antibiotic use in India has been linkedto increased antibiotic resistance [32]. Resistance to antibiotics is problematic even in the treatmentof keratitis, where a topical application of antibiotics is used. Infection with antibiotic resistant

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strains results in prolonged infection [33], more severe keratitis [5], and an increase in the cost oftreatment [34,35].

Indian P. aeruginosa strains harbored more resistance genes compared to Australian isolates,although aph(3’)-IIb, blaPAO1 (fosA), and catB7 were found in all isolates, which was consistentwith previous studies [31,36]. qnrVC1 was found in four Indian isolates but no Australian isolates.This fluoroquinolone resistance gene has not been previously reported in P. aeruginosa ocular isolates [31],but it has been reported in burns isolates and has been identified as being carried on an integron [37].Similarly, in the current study qnrVC1 was carried on a class 1 integron in isolates 202 and 221,but integrated into a Tn3 transposon in isolates 198 and 219. This gene has also been isolated from thehigh risk ST773 clone of P. aeruginosa from urine in Hungary [38]. High risk clones are isolates withhigh mutational rates in resistance genes and those that have acquired a large number of resistancegenes. As previously described, resistance to fluoroquinolones in keratitis P. aeruginosa isolates was alsodue to mutations in the quinolone resistance determining regions of gyrA and parC [15]. Possession ofqnrVC1 and mutations in gyrA and parC were associated with high levels of fluroquinolone resistance.The possession of large numbers of acquired resistance genes by Indian isolates likely contributed tothe higher rates of resistance of these isolates. The Indian isolates 198, 202, 217, 219, 220, and 221 alsohad a high number of gene variations which is an independent mechanism of resistance.

The aminoglycoside resistance gene aph(6)-Id which encodes for streptomycin resistance was foundin six Indian isolates, including the four that carried qnrVC1, but in no Australian isolates. Previously,aph(6)-Id was found in only one Indian ocular isolate from 1997 [31], but has been found in cystic fibrosisP. aeruginosa isolates [39] and has been associated with the transposon Tn5393 on a plasmid in onestrain of P. aeruginosa [39]. As streptomycin is no longer used in clinical treatment [40], this resistancemay not be clinically relevant but does suggest environmental selection for the persistence of this gene.

The total number of gene variants found in the Indian isolates 198, 202, 219, and 221 weregreater than Australian isolates. However, there were a small number of SNPs found in the genesassociated with resistance for these isolates. The Indian isolate 206 (NEWST) had a high number ofSNPs in antibiotic resistance genes mexC, mexD, mexM, mexX, mexS, opmE, mexP, mexK, oprJ, ampC,rosC, and mprF. There was no difference in the mutations of other mex genes including mexX, mexT,mexD, mexM, and mexY between Australian and Indian isolates. Given that most isolates from bothcountries, whether they were sensitive or resistant, had a similar number of mutations in the resistancegenes, it is likely that the resistance to antibiotics was related to the possession of acquired resistancegenes rather than mutations in chromosomal genes.

In the Australian isolates, four out of the eight isolates (50%) carried exoU, while one isolate wasboth exoU+/exoS+ and three (38%) were exoS+. In Indian isolates, 50% carried exoU and 50% carried exoS.A previous study has also shown an equal ratio of both genes [41] in keratitis isolates. The possession of theexoU genotype in P. aeruginosa ocular isolates has been related to elevated resistance to disinfectants [42],fluoroquinolones [43], and multiple antibiotics [41]. Furthermore, one study reported worst clinicaloutcomes and more resistance by exoU carrying isolates [43]. The isolates of this study showed similarfindings because the exoU+ isolates 198, 202, 217, 219, 220, 221, and 233 were also MDR.

The DNA mismatch repair system (MMR) in P. aeruginosa is based on the protein trimerMutS-MutL-UvrD and functions to correct errors and preserve the integrity of the genome [24,44,45].The mutH component of MMR, which is important in other Gram negative bacteria, such as E. coli [46],has not been found previously in P. aeruginosa [25] and was not present in the isolates of the current study.Mutations in mutS, mutL and uvrD can reduce the ability of the bacterium to repair DNA lesions [46].Strong mutator strains have defects in their MMR system and mutations in mutS predominate [47].Mutations in the MMR can be a reason for the development of hypermutations in isolates. In cysticfibrosis, hypermutations were found to be a key factor in the development of MDR resistant P. aeruginosastrains [23]. Similar findings were found in this study where isolates 198, 202, 206, 219, and 221 hadmutations in the MMR genes, and these isolates had an overall larger variation in their genomes.In the current study, isolates had more mutations in mutL and uvrD, suggesting the strains may not be

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strong mutators (which is usually associated with mutation in mutS), but nevertheless can undergouncorrected genetic changes. Indeed, the P. aeruginosa isolates in the current study which had mutationsin mutL and uvrD had greater numbers of SNPs, insertions and deletions, acquired genes, and had largepan genomes. Among these isolates, 198, 202, 219, and 221 possessed either the transposon Tn3 or class1 integrons which carried the acquired genes. This also might be due to mutated MMR, as mutationsin MMR genes increase the chances of horizontal gene transfer in mutator isolates [47]. The number ofmutations in MMR was greater in exoU possessing isolates with large gene variations. exoU is carriedon genomic islands [48,49] and these exoU carrying isolates had larger pan genomes with possession ofmobile genetic elements. Therefore, the isolates with the mutated MMR systems may have a greaterability of strains to accumulate gene variations and the acquisition of exoU. Isolate 206, on the otherhand, possessed exoS and was not MDR but possessed a large number of SNPs and a large pan genomewith one mutation in each of mutS and mutL. Further in-depth studies are required to understand theinfluence of the MMR system on genomic changes in P. aeruginosa.

Analysis of the sequence types of the P. aeruginosa ocular isolates revealed the presence of threeclones, two in the Indian and one in the Australian isolates. The isolates with the same STs had mostlythe same phenotypic and genotypic features. The exception to this was isolate 220 that had acquiredfewer resistance genes compared to the other two isolates 202 and 221 of ST316. Previously, five ocularP. aeruginosa strains from India isolated in 1997 were of sequence type ST308 [31]. The two isolates ofST308 in the current study, isolated in 2017 and 2018, had acquired more resistance genes comparedto isolates from 1997 [31]. This indicates that the clonal isolates have continued to evolve over thistime period, although the specific selection factors driving those changes are yet to be elucidated.None of the isolates were collected from the same patient. The majority of the isolates with the sameSTs grouped in the same phylogeny including previously published isolates (PA_D1, PA_D2, PA_D9,P_D16) with ST1971.

Core and pan genome phylogenies of the isolates produced two almost identical groups, whichwas in agreement with previously published studies [31,50]. Both phylogenies included isolates fromeither Australia or India, but those in group 2 tended to be the MDR Indian isolates and possessedhigher numbers of antibiotic resistance genes. About 65% of all ocular isolates grouped together whichindicated less diversity in the ocular P. aeruginosa isolates [31,51,52]. The grouping of MDR strainsfrom this study with PA14 along a MDR ocular isolate VRFPA04 [36] in both core genome and pangenome analysis, and the grouping of the sensitive strains with PAO1 along the commonly studiedcystic fibrosis isolates DK2 and LESB58, was similar to a previous study examining older isolatesfrom India and Australia [31]. Isolate 206, which had the smallest number of core genes and was ofa new sequence type, was an outlier in the core genome phylogeny similar to the taxonomic outlierPA7 [53]. However, isolate 206 was grouped together with other isolates in the pan genome becauseit had acquired a large number of genes. Acquired genes are part of the pan rather than the coregenome [53] and the presence of larger pan genomes in MDR P. aeruginosa isolates points towards theacquisition of new genes [54]. Previously, a smaller core genome size of 4910 genes has been reportedin ocular P. aeruginosa isolates [31]. However, the current study found a core genome size similar toP. aeruginosa from different sources, comprising 5316–5233 genes [55,56]. The core genome (which isalmost 90% of total genome) refers to the conserved genes present in a species [57] which might differin each individual strain within that species. Additionally, SNPs can be a result of poor sequencingquality and hence it is important to have a good sequencing depth at those positions to identify themas a mutation rather than sequencing error [58]. Grouping of all the isolates including ocular andnon-ocular remained the same in both core and pan genome phylogeny.

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4. Materials and Methods

4.1. P. aeruginosa Strains and Susceptibility Testing

Twenty-six P. aeruginosa keratitis isolates, eight isolated in Australia from 2004 to 2006, six from2018 and 2019 (total 14 Australian isolates), and twelve isolated in India between 2017 and 2018, wereincluded in this study. These isolates were selected from a larger collection of strains based on theirantibiotic susceptibilities (those phenotypically resistant to multiple antibiotics, some resistant to one ormultiple antibiotics, and some which were sensitive to all antibiotics). The susceptibilities of Australianstrains (2004–2006) included in this study have been previously published [29]. Strains were selectedafter comparing their susceptibilities to antibiotics that are used to treat ocular infections. For geneticcomparisons, the data of 34 P. aeruginosa isolates from eyes and other sources were also included.The general characteristics of these isolates are described in Supplementary Table S4. The genomesof these isolates were downloaded from the NCBI database and reannotated for this study using thesame parameters as of the isolates of this study to avoid any bias in results.

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) ofvarious antibiotics which are commonly used to treat P. aeruginosa keratitis [16] were assessed for theisolates using the broth microdilution method in 96-well plates following the Clinical and LaboratoryStandard Institute guidelines [59]. The antibiotics tested were ciprofloxacin, levofloxacin, gentamicin,ceftazidime (Sigma-Aldrich, St. Louis, MO, USA), polymyxin B (Sigma-Aldrich, Vandtårnsvej, Søborg,Denmark), tobramycin, piperacillin (Cayman Chemical Company, Ann Arbor, MI, USA), and imipenem(LKT Laboratories Inc., St. Paul, MN, USA). The susceptibility results were interpreted using theEUCAST v9 [60] and CLSI [61] 2017 breakpoints.

4.2. Genomic Sequencing

DNeasy Blood and Tissue Kits (Qiagen, Hilden, Germany) were used for DNA extraction asper the manufacturer’s recommendations. The Nextera XT DNA library preparation kit (Illumina,San Diego, CA, USA) was used to prepare paired-end libraries. All the libraries were multiplexed onone MiSeq run. FastQC version 0.117 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc) wasused to assess the quality of sequenced genomes using raw reads. Version 0.38 of Trimmomatic [61] wasused for trimming the adapters from the reads following de novo assembly using Spades v3.13.0 [62].Genomes were annotated using Prokka v1.12 [63].

Sequence types were investigated using PubMLST https://pubmlst.org/. Pan genomes of theP. aeruginosa isolates were analyzed using Roary v3.11.2 [64] using PAO1 as a reference, while coregenome phylogeny was constructed using Harvest Suite Parsnp v1.2 [65] with strains PAO1, PA7,and PA14 used as reference strains. The output file ‘genes_ presence_absence’ was used to compare theP. aeruginosa isolates. Acquired resistance genes were identified using the online database Resfinder v3.1(Centre for Genomic Epidemiology, DTU, Denmark) [66]. Integron Finder v1.5.1 was used to identifyany integrons present in the isolates. Mutations in the genes were detected using Snippy V2 [67].Isolates with same sequence types were compared for nucleotide similarities using the MUMmeronline web tool (http://jspecies.ribohost.com/jspeciesws/#analyse).

Using the Pseudomonas genome database (http://www.pseudomonas.com) and comprehensiveantibiotic resistance database (https://card.mcmaster.ca), 76 genes related to P. aeruginosa resistance wereselected to investigate the presence of single nucleotide polymorphisms. All isolates were analyzedfor the presence of the type III secretion system associated virulence factors exoU and exoS using theBlastN database.

4.3. Statistical Analysis

The statistical analysis was performed using GraphPad Prism v8. Medians were calculated withthe ‘descriptive statistics’ option during analysis of variance (ANOVA). P-values less than 0.05 wereconsidered as significant. Fischer’s Exact test was used to find the difference between acquired genes.

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To analyze the significant difference in the DNA mismatch repair genes between exoU and exoS isolatesand gene variations in the isolates, the Mann–Whitney test was used.

5. Conclusions

Indian isolates and Australian isolates were clearly distinct in carrying a type III secretion systemrelated to exoU and exoS. There was an association in the isolates for carrying acquired resistance geneswith a large number of pan genes. Indian isolates were more resistant to antibiotics compared toAustralian isolates. Additionally, isolates of P. aeruginosa from ocular infection had a large number ofgenetic variations (mutations) and a mutated mismatch repair system. However, the isolates collectedfrom the same region or time will give a clearer idea of these differences.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/9/600/s1,Table S1: Details of the Pseudomonas aeruginosa isolates used in the current study. Table S2: Gene variationsof resistance genes in Pseudomonas aeruginosa isolates (the gray-shaded strains were isolated from Australia).Nucleotide accession: The nucleotide sequences are available in the GenBank under the Bio project accessionnumber PRJNA590804. Table S3: Types of mutations in the mismatch repair system; Table S4: Genomics featuresof P. aeruginosa isolates; Figure S1: Pan-genome phylogenetic tree. The data on the right of the figure shows thepresence and absence of genes. The tree was built using the genome of PAO1 as a reference.

Author Contributions: Conceptualization, M.K., F.S., and M.D.P.W.; methodology, M.K., F.S., S.S., S.A.R., andM.D.P.W.; writing—original draft preparation, M.K.; writing—review and editing, F.S., S.A.R., and M.D.P.W.;supervision, F.S. and M.D.P.W.; funding acquisition, F.S. All authors have read and agreed to the published versionof the manuscript.

Funding: This research received no external funding.

Acknowledgments: The authors would like to acknowledge the Singapore Centre for Environmental Life SciencesEngineering (SCELSE), whose research is supported by the National Research Foundation Singapore, Ministry ofEducation, Nanyang Technological University, and National University of Singapore, under its Research Centre ofExcellence Programme. We would also like to acknowledge Engineer Ahsan Ullah Khan, Independent MonitoringUnit Haripur KPK, Pakistan, for his help in the computational analysis and Doctor Nicole Carnt, School ofOptometry and Vision Science UNSW, Sydney, for her help in the isolates collection. We are also thankful toUNSW high performance computing facility KATANA for providing us with the cluster time for the data analysis.

Conflicts of Interest: The authors declare no conflict of interest.

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